Are aptamer-based biosensing approaches a good choice for female fertility monitoring? A comprehensive review.

The WHO estimates that 8-10% of couples are facing fertility problems, often due to inaccuracy in predicting the female's ovulation period controlled by four key hormones. The quantification and monitoring of such key hormones are crucial for the early identification of infertility, but also in improving therapeutic management associated with hormonal imbalance. In this review, we extensively summarize and discuss: i) drawbacks of laboratory methods for fertility testing (costly, invasive, complex) and commercially available point-of-care tests (measuring only one/two of the four key hormones), ii) the understanding of different biosensors for fertility monitoring, and iii) an in-depth classification and overview of aptamer-based sensing of the hormones of interest. This review provides insights on hormone detection strategies for fertility, with a focus on the classification of the current 'aptasensing' strategies, aiming to assist as a basic guide for the development of accurate fertility window monitoring tools based on aptamers.

Development of a combi-electrosensor for the detection of phenol by combining photoelectrochemistry and square wave voltammetry.

The high toxicity, endocrine-disrupting effects and low (bio)degradability commonly attributed to phenolic compounds have promoted their recognition as priority toxic pollutants. For this reason, the monitoring of these compounds in industrial, domestic and agricultural streams is crucial to prevent and decrease their toxicity in our daily life. To confront this relevant environmental issue, we propose the use of a combi-electrosensor which combines singlet oxygen (1O2)-based photoelectrochemistry (PEC) with square wave voltammetry (SWV). The high sensitivity of the PEC sensor (being a faster alternative for traditional chemical oxygen demand-COD-measurements) ensures the detection of nmol L-1 levels of phenolic compounds while the SWV measurements (being faster than the color test kits) allow the differentiation between phenolic compounds. Herein, we report on the development of such a combi-electrosensor for the sensitive and selective detection of phenol (PHOH) in the presence of related phenolic compounds such as hydroquinone (HQ), bisphenol A (BPA), resorcinol (RC) and catechol (CC). The PEC sensor was able to determine the concentration of PHOH in spiked river samples containing only PHOH with a recovery between 96% and 111%. The SWV measurements elucidated the presence of PHOH, HQ and CC in the spiked samples containing multiple phenol compounds. Finally, the practicality of the combi-electrosensor set-up with a dual SPE containing two working electrodes and shared reference and counter electrodes was demonstrated. As a result, the combination of the two techniques is a powerful and valuable tool in the analysis of phenolic samples, since each technique improves the general performance by overcoming the inherent drawbacks that they display independently.

Novel electrochemiluminescent assay for the aptamer-based detection of testosterone.

This work presents a proof-of-concept assay for the detection and quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding (TESS.1) aptamer was used to demonstrate the novel methodology. Upon binding of the target, the TESS.1 aptamer is released from its complementary capture probe - previously immobilized at the surface of the electrode - producing a decrease in the ECL signal after a washing step removing the released (labeled) TESS.1 aptamer. The analytical capability of the ECL assay towards testosterone detection was investigated displaying a linear range from 0.39 to 1.56 µM with a limit of detection of 0.29 µM. The selectivity of the proposed assay was assessed by performing two different negative control experiments; i) detection of testosterone with a randomized ssDNA sequence and ii) detection of two other steroids, i.e. deoxycholic acid and hydrocortisone with the TESS.1 aptamer. In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of the binding, and to characterize aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (Kd) of the complex of testosterone and the TESS.1 aptamer. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. Furthermore, this important characterization carried out in parallel validates the real functionality of the aptamer (TESS.1) ensuring its use towards selective testosterone binding in further biosensors. This research will pave the way for the development of new aptamer-based assays coupled with ECL sensing for the detection of relevant small molecules.

Modulating the mixed potential for developing biosensors: Direct potentiometric determination of glucose in whole, undiluted blood.

The growing demand for tools to generate chemical information in decentralized settings is creating a vast range of opportunities for potentiometric sensors, since their combination of robustness, simplicity of operation and cost can hardly be rivalled by any other technique. In previous works, we have shown that the mixed potential of a Pt electrode can be controlled with analytical purposes using a coating of Nafion, thus providing a way to develop a potentiometric biosensor for glucose. Unfortunately, the linear range of this device did not match the relevant clinical range for glucose in blood. This work presents a novel strategy to control the mixed potential that allows the development of a potentiometric biosensor for the direct detection of glucose in whole, undiluted blood without any sample pretreatment. By changing the ionomer, the analytical response can be tuned, shifting the linear range while keeping the sensitivity. Aquivion, a polyelectrolyte from the same family as Nafion, is used to stabilize the mixed potential of a platinized paper-based electrode, to entrap the enzyme and to reduce the interference from negatively charged species. Factors affecting the generation of the signal and the principle of detection are discussed. Optimization of the biosensor composition was achieved with particular focus on the characterization of the linear range and sensitivity. The accurate measurement of blood sugar levels in a single drop of whole blood with excellent recovery is presented.

Thin-Layer Potentiometry for Creatinine Detection in Undiluted Human Urine Using Ion-Exchange Membranes as Barriers for Charged Interferences.

Herein, thin-layer potentiometry combined with ion-exchange membranes as barriers for charged interferences is demonstrated for the analytical detection of creatinine (CRE) in undiluted human urine. Briefly, CRE diffuses through an anion-exchange membrane (AEM) from a sample contained in one fluidic compartment to a second reservoir, containing the enzyme CRE deiminase. There, CRE reacts with the enzyme, and the formation of ammonium is dynamically monitored by potentiometric ammonium-selective electrodes. This analytical concept is integrated into a lab-on-a-chip microfluidic cell that allows for a high sample throughput and the operation under stop-flow mode, which allows CRE to passively diffuse across the AEM. Conveniently, positively charged species (i.e., potassium, sodium, and ammonium, among others) are repelled by the AEM and never reach the ammonium-selective electrodes; thus, possible interference in the response can be avoided. As a result, the dynamic potential response of the electrodes is entirely ascribed to the stoichiometric formation of ammonium. The new CRE biosensor exhibits a Nernstian slope, within a linear range of response from 1 to 50 mM CRE concentration. As expected, the response time (15-60 min) primarily depends on the CRE diffusion across the AEM. CRE analysis in urine samples displayed excellent results, without requiring sample pretreatment (before the introduction of the sample in the microfluidic chip) and with high compatibility with development into a potential point-of-care clinical tool. In an attempt to decrease the analysis time, the presented analytical methodology for CRE detection is translated into an all-solid-state platform, in which the enzyme is immobilized on the surface of the ammonium-selective electrode and with the AEM on top. While more work is necessary in this direction, the CRE sensor appears to be promising for CRE analysis in both urine and blood.

Controlling the mixed potential of polyelectrolyte-coated platinum electrodes for the potentiometric detection of hydrogen peroxide.

The use of a Pt electrode coated with a layer of Nafion has been described in previous works as an attractive way to perform the potentiometric detection of hydrogen peroxide. Despite of the attractive features of this approach, the nature of the non-Nernstian response of this system was not properly addressed. In this work, using a mixed potential model, the open circuit potential of the Pt electrode is shown to be under kinetic control of the oxygen reduction reaction (ORR). It is proposed that hydrogen peroxide acts as an oxygenated species that blocks free sites on the Pt surface, interfering with the ORR. Therefore, the effect of the polyelectrolyte coating can be understood in terms of the modulation of the factors that affects the kinetics of the ORR, such as an increase of the H+ concentration, minimization of the effect of the spectator species, etc. Because of the complexity and the lack of models that accurately describe systems with practical applications, this work is not intended to provide a mechanistic but rather a phenomenological view on problem. A general framework to understand the factors that affect the potentiometric response is provided. Experimental evidence showing that the use of polyelectrolyte coatings are a powerful way to control the mixed potential open new ways for the development of robust and simple potentiometric sensors.

The sweet detection of rolling circle amplification: Glucose-based electrochemical genosensor for the detection of viral nucleic acid.

Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 µg mL-1 of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.

Cytotoxicity Study of Ionophore-Based Membranes: Toward On-Body and in Vivo Ion Sensing.

We present the most complete study to date comprising in vitro cytotoxicity tests of ion-selective membranes (ISMs) in terms of cell viability, proliferation, and adhesion assays with human dermal fibroblasts. ISMs were prepared with different types of plasticizers and ionophores to be tested in combination with assays that focus on the medium-term and long-term leaching of compounds. Furthermore, the ISMs were prepared in different configurations considering (i) inner-filling solution-type electrodes, (ii) all-solid-state electrodes based on a conventional drop-cast of the membrane, (iii) peeling after the preparation of a wearable sensor, and (iv) detachment from a microneedle-based sensor, thus covering a wide range of membrane shapes. One of the aims of this study, other than the demonstration of the biocompatibility of various ISMs and materials tested herein, is to create an awareness in the scientific community surrounding the need to perform biocompatibility assays during the very first steps of any sensor development with an intended biomedical application. This will foster meeting the requirements for subsequent on-body application of the sensor and avoiding further problems during massive validations toward the final in vivo use and commercialization of such devices.

Wearable Potentiometric Ion Patch for On-Body Electrolyte Monitoring in Sweat: Toward a Validation Strategy to Ensure Physiological Relevance.

Herein, the reproducibility and a double validation of on-body measurements provided by new wearable potentiometric ion sensors (WPISs) is presented. Sweat collected during sport practice was first analyzed using the developed device, the pH-meter, and ion chromatography (IC) prior to on-body measurements (off-site validation). Subsequently, the accuracy of on-body measurements accomplished by the WPISs was evaluated by comparison with pH-meter readings and IC after collecting sweat (every 10-12.5 min) during sport practice. The developed device contains sensors for pH, Cl-, K+, and Na+ that are embedded in a flexible sampling cell for sweat analysis. The electrode array was fabricated employing MWCNTs (as an ion-to-electron transducer) and stretchable materials that have been exhaustively characterized in terms of analytical performance, presenting Nernstian slopes within the expected physiological range of each ion analyte (Cl-, 10-100 mM; K+, 10-10 mM; and Na+, 10-100 mM and pH, 4.5-7.5), drift suitable for midterm exercise practice (0.3 ± 0.2 mV h-1), fast response time, adequate selectivity for sweat measurements, and excellent reversibility. Besides that, the designed sampling cell avoids any sweat contamination and evaporation issues while supplying a passive sweat flow encompassing specifically the individual's perspiration. The interpretation of ion concentration profiles may permit the identification of personal dynamic patterns in sweat composition while practicing sport.

Modern creatinine (Bio)sensing: Challenges of point-of-care platforms.

The importance of knowing creatinine levels in the human body is related to the possible association with renal, muscular and thyroid dysfunction. Thus, the accurate detection of creatinine may indirectly provide information surrounding those functional processes, therefore contributing to the management of the health status of the individual and early diagnosis of acute diseases. The questions at this point are: to what extent is creatinine information clinically relevant?; and do modern creatinine (bio)sensing strategies fulfil the real needs of healthcare applications? The present review addresses these questions by means of a deep analysis of the creatinine sensors reported in the literature over the last five years. There is a wide range of techniques for detecting creatinine, most of them based on optical readouts (20 of the 33 papers collected in this review). However, the use of electrochemical techniques (13 of the 33 papers) is recently emerging in alignment with the search for a definitive and trustworthy creatinine detection at the point-of-care level. In this sense, biosensors (7 of the 33 papers) are being established as the most promising alternative over the years. While creatinine levels in the blood seem to provide better information about patient status, none of the reported sensors display adequate selectivity in such a complex matrix. In contrast, the analysis of other types of biological samples (e.g., saliva and urine) seems to be more viable in terms of simplicity, cross-selectivity and (bio)fouling, besides the fact that its extraction does not disturb individual's well-being. Consequently, simple tests may likely be used for the initial check of the individual in routine analysis, and then, more accurate blood detection of creatinine could be necessary to provide a more genuine diagnosis and/or support the corresponding decision-making by the physician. Herein, we provide a critical discussion of the advantages of current methods of (bio)sensing of creatinine, as well as an overview of the drawbacks that impede their definitive point-of-care establishment.

A novel wireless paper-based potentiometric platform for monitoring glucose in blood.

A novel low-cost, compact and sensitive paper-based platform for the accurate monitoring of glucose in biological fluids is presented. Paper-based working and reference electrodes are combined to build a whole potentiometric cell, which also fits a sampling module for simple and fast determination of glucose in a single drop of blood. The working electrode is built using a platinized filter paper coated with a Nafion membrane that entraps the enzyme glucose oxidase; the reference electrode is made by casting a polyvinylbutyral-based membrane onto a conductive paper. The system works by detecting the hydrogen peroxide generated as a result of the enzymatic reaction. Selectivity is achieved due to the permselective behaviour of Nafion, while a significant enhancement of the sensitivity is reached by exploiting the Donnan-coupled formal potential. Under optimum conditions, a sensitivity of -95.9 ± 4.8 mV per decade in the 0.3-3 mM range is obtained. Validation of the measurements has been performed against standard methods in human serum and blood. Final integration with a wireless reader allows for truly in situ measurements with a less than 2 minute procedure including a two-point calibration, washing and measurement. This low-cost analytical device opens up new prospects for rapid diagnostic results in non-laboratory settings.

Paper-based enzymatic electrode with enhanced potentiometric response for monitoring glucose in biological fluids.

A novel paper-based potentiometric sensor with an enhanced response for the detection of glucose in biological fluids is presented. The electrode consists on platinum sputtered on a filter paper and a Nafion membrane to immobilize the enzyme glucose oxidase. The response obtained is proportional to the logarithm of the concentration of glucose, with a sensitivity of -119±8mV·decade-1, a linear range that spans from 10-4M to 10-2.5 M and a limit of detection of 10-4.5 M of glucose. It is shown that Nafion increases the sensitivity of the technique while minimizing interferences. Validation with human serum samples shows an excellent agreement when compared to standard methods. This approach can become an interesting alternative for the development of simple and affordable devices for point of care and home-based diagnostics.

A Textile-Based Stretchable Multi-Ion Potentiometric Sensor.

A textile-based wearable multi-ion potentiometric sensor array is described. The printed flexible sensors operate favorably under extreme mechanical strains (that reflect daily activity) while offering attractive real-time noninvasive monitoring of electrolytes such as sodium and potassium.